Application of droplet digital PCR in minimal residual disease monitoring of rare fusion transcripts and mutations in haematological malignancies.

Leukaemia of various subtypes are driven by distinct chromosomal rearrangement or genetic abnormalities. The leukaemogenic fusion transcripts or genetic mutations serve as molecular markers for minimal residual disease (MRD) monitoring. The current study evaluated the applicability of several droplet digital PCR assays for the detection of these targets at RNA and DNA levels (atypical BCR::ABL1 e19a2, e23a2ins52, e13a2ins74, rare types of CBFB::MYH11 (G and I), PCM1::JAK2, KMT2A::ELL2, PICALM::MLLT10 fusion transcripts and CEBPA frame-shift and insertion/duplication mutations) with high sensitivity. The analytical performances were assessed by the limit of blanks, limit of detection, limit of quantification and linear regression. Our data demonstrated serial MRD monitoring for patients at molecular level could become "digitalized", which was deemed important to guide clinicians in treatment decision for better patient care.

Distinct karyotypic and mutational landscape in trisomy AML.

Trisomy karyotype occurs in 5%-10% of AML. Its mutational landscape and prognostic significance are not well defined. A cohort of 156 trisomy AML patients was analysed, with reference to 615 cytogenetically normal (CN) AML patients. Trisomy AML showed distinct mutational landscape with more prevalent SMC1A, N/KRAS, ASXL1 and BCOR but fewer CEBPAbZIP and NPM1 mutations in patients ≤60, and fewer NPM1 mutations in those >60. NRAS mutations were associated with poor outcome in trisomy AML, whereas DNMT3A and FLT3-ITD mutations had neutral effect. Trisomy AML appeared biologically distinct from CN-AML.

The impact of the multi-disciplinary molecular tumour board and integrative next generation sequencing on clinical outcomes in advanced solid tumours.

Background: The integration of next-generation sequencing (NGS) comprehensive gene profiling (CGP) into clinical practice is playing an increasingly important role in oncology. Therefore, the HKU-HKSH Multi-disciplinary Molecular Tumour Board (MTB) was established to advance precision oncology in Hong Kong. A multicenter retrospective study investigated the feasibility of the HKU-HKSH MTB in determining genome-guided therapy for treatment-refractory solid cancers in Hong Kong. Methods: Patients who were presented at the HKU-HKSH MTB between August 2018 and June 2022 were included in this study. The primary study endpoints were the proportion of patients who receive MTB-guided therapy based on genomic analysis and overall survival (OS). Secondary endpoints included the proportion of patients with actionable genomic alterations, objective response rate (ORR), and disease control rate (DCR). The Kaplan-Meier method was used in the survival analyses, and hazard ratios were calculated using univariate Cox regression. Findings: 122 patients were reviewed at the HKU-HKSH MTB, and 63% (n = 77) adopted treatment per the MTB recommendations. These patients achieved a significantly longer median OS than those who did not receive MTB-guided therapy (12.7 months vs. 5.2 months, P = 0.0073). Their ORR and DCR were 29% and 65%, respectively. Interpretation: Our study demonstrated that among patients with heavily pre-treated advanced solid cancers, MTB-guided treatment could positively impact survival outcomes, thus illustrating the applicability of NGS CGPs in real-world clinical practice. Funding: The study was supported by the Li Shu Pui Medical Foundation. Dr Aya El Helali was supported by the Li Shu Pui Medical Foundation Fellowship grant from the Li Shu Pui Medical Foundation. Funders had no role in study design, data collection, data analysis, interpretation, or writing of the report.

Effect on Germline Mutation Rate in a High-Risk Chinese Breast Cancer Cohort after Compliance with The National Comprehensive Cancer Network (NCCN) 2023 v.1 Testing Criteria.

BACKGROUND: The National Comprehensive Cancer Network (NCCN) testing criteria for the high-penetrance breast cancer susceptibility genes, specifically BRCA1, BRCA2, CDH1, PALB2, PTEN, and TP53, have been recently modified in 2023 to 2023 v.1. The following criteria have been changed: (1) from a person diagnosed with breast cancer at ≤45 to ≤50; (2) from aged 45-50 of personal breast diagnosis to any age of diagnosis with multiple breast cancers; and (3) from aged ≥51 of personal breast diagnosis to any age of diagnosis with family history listed in NCCN 2022 v.2. METHODS: High-risk breast cancer patients (n = 3797) were recruited from the Hong Kong Hereditary Breast Cancer Family Registry between 2007 and 2022. Patients were grouped according to NCCN testing criteria 2023 v.1 and 2022 v.2. A 30-gene panel for hereditary breast cancer was performed. The mutation rates on high-penetrance breast cancer susceptibility genes were compared. RESULTS: About 91.2% of the patients met the 2022 v.2 criteria, while 97.5% of the patients met the 2023 v.1 criteria. An extra 6.4% of the patients were included after the revision of the criteria, and 2.5% of the patients did not meet both testing criteria. The germline BRCA1/2 mutation rates for patients meeting the 2022 v.2 and 2023 v.1 criteria were 10.1% and 9.6%, respectively. The germline mutation rates of all 6 high-penetrance genes in these two groups were 12.2% and 11.6%, respectively. Among the additional 242 patients who were included using the new selection criteria, the mutation rates were 2.1% and 2.5% for BRCA1/2 and all 6 high-penetrance genes, respectively. Patients who did not meet both testing criteria were those with multiple personal cancers, a strong family history of cancers not listed in the NCCN, unclear pathology information, or the patient's voluntary intention to be tested. The mutation rates of BRCA1/2 and the 6 high-penetrance genes in these patients were 5.3% and 6.4%, respectively. CONCLUSION: This study provided a real-world application of the revision of NCCN guidelines and its effect on the germline mutation rate in the Chinese population. Applying the updated criteria for further genetic investigation would increase the positive detection rate, and potentially more patients would benefit. The balance between the resource and outcome requires careful consideration.

Acute promyelocytic leukaemia: population-based study of epidemiology and outcome with ATRA and oral-ATO from 1991 to 2021.

BACKGROUND: The epidemiology and treatment of acute promyelocytic leukaemia (APL) are changing. We have incorporated oral arsenic trioxide (oral-ATO) into induction/maintenance. METHODS: Newly-diagnosed APL from 1991 to 2021 divided into three 10-year periods were studied to define its epidemiology and how oral-ATO impacted on its outcome. Primary endpoints included APL incidence, early deaths (ED, first 30 days), and overall survival (OS). Secondary endpoints included post-30-day OS, relapse-free survival (RFS), and incidence of second cancers. RESULTS: APL occurred in 374 males and 387 females at a median age of 44 (1-97) years. Annual incidences increased progressively, averaging 0.32 per 100,000 people. All-trans retinoic acid (ATRA)-based and oral-ATO-based regimens were used in 469 and 282 patients. There were 144 EDs, occurring almost exclusively in ATRA-based inductions (N = 139), being more with males, age > 50 years, leucocyte > 10 × 109/L, diagnosis during 1991-2009 and fewer with oral-ATO-based regimens. After a median of 75 (interquartile range: 14-161) months, 5-year and 10-year OS were 68.1% and 63.3%, inferior with males, age > 50 years, leucocyte > 10 × 109/L, high-risk Sanz score and superior with oral-ATO-based regimens. Factoring out EDs, 5-year and 10-year post-30-day OS were 84.0% and 78.1%, inferior with males and superior with oral-ATO-based regimens. In 607 CR1 patients, the 5-year RFS was 83.8%, superior with diagnosis in 2010-2021 and oral-ATO-based regimens. Second cancers developed in 21 patients, unrelated to oral-ATO-based regimens. CONCLUSIONS: There was an increasing incidence of APL, and all survivals were superior with the use of oral-ATO-based regimens. This study formed part of the Acute Promyelocytic Leukaemia Asian Consortium Project (ClinicalTrials.gov identifier: NCT04251754).

Genetic Testing and Its Clinical Application in Prostate Cancer Management: Consensus Statements from the Hong Kong Urological Association and Hong Kong Society of Uro-Oncology.

Background: In recent years, indications for genetic testing in prostate cancer (PC) have expanded from patients with a family history of prostate and/or related cancers to those with advanced castration-resistant disease, and even to early PC patients for determination of the appropriateness of active surveillance. The current consensus aims to provide guidance to urologists, oncologists and pathologists working with Asian PC patients on who and what to test for in selected populations. Methods: A joint consensus panel from the Hong Kong Urological Association and Hong Kong Society of Uro-Oncology was convened over a series of 5 physical and virtual meetings. A background literature search on genetic testing in PC was performed in PubMed, ClinicalKey, EBSCOHost, Ovid and ProQuest, and three working subgroups were formed to review and present the relevant evidence. Meeting agendas adopted a modified Delphi approach to ensure that discussions proceed in a structured, iterative and balanced manner, which was followed by an anonymous voting on candidate statements. Of 5 available answer options, a consensus statement was accepted if ≥ 75% of the panelists chose "Accept Completely" (Option A) or "Accept with Some Reservation" (Option B). Results: The consensus was structured into three parts: indications for testing, testing methods, and therapeutic implications. A list of 35 candidate statements were developed, of which 31 were accepted. The statements addressed questions on the application of PC genetic testing data and guidelines to Asian patients, including patient selection for germline testing, selection of gene panel and tissue sample, provision of genetic counseling, and use of novel systemic treatments in metastatic castration-resistant PC patients. Conclusion: This consensus provides guidance to urologists, oncologists and pathologists working with Asian patients on indications for genetic testing, testing methods and technical considerations, and associated therapeutic implications.

Comparison of novel rapid diagnostic of blood culture identification and antimicrobial susceptibility testing by Accelerate Pheno system and BioFire FilmArray Blood Culture Identification and BioFire FilmArray Blood Culture Identification 2 panels.

BACKGROUND: Conventional turnaround time (TAT) for positive blood culture (PBC) identification (ID) and antimicrobial susceptibility testing (AST) is 2-3 days. We evaluated the TAT and ID/AST performance using clinical and seeded samples directly from PBC bottles with different commercial approaches: (1) Accelerate Pheno® system (Pheno) for ID/AST; (2) BioFire® FilmArray® Blood Culture Identification (BCID) Panel and/ or BCID2 for ID; (3) direct AST by VITEK® 2 (direct AST); and (4) overnight culture using VITEK® 2 colony AST. RESULTS: A total of 141 PBC samples were included in this evaluation. Using MALDI-TOF (Bruker MALDI Biotyper) as the reference method for ID, the overall monomicrobial ID sensitivity/specificity are as follows: Pheno 97.9/99.9%; BCID 100/100%; and BCID2 100/100%, respectively. For AST performance, broth microdilution (BMD) was used as the reference method. For gram-negatives, overall categorical and essential agreements (CA/EA) for each method were: Pheno 90.3/93.2%; direct AST 92.6/88.5%; colony AST 94.4/89.5%, respectively. For gram-positives, the overall CA/EAs were as follows: Pheno 97.2/98.89%; direct AST 97.2/100%; colony AST 97.2/100%, respectively. The BCID/BCID2 and direct AST TATs were around 9-20 h (1/9-19 h for ID with resistance markers/AST), with 15 min/sample hands-on time. In comparison, Pheno TATs were around 8-10 h (1.5/7 h for ID/AST) with 2 min/sample hands-on time, maintains a clinically relevant fast report of antibiotic minimal inhibitory concentration (MIC) and allows for less TAT and hands-on time. CONCLUSION: In conclusion, to the best of our knowledge, this is the first study conducted as such in Asia; all studied approaches achieved satisfactory performance, factors such as TAT, panel of antibiotics choices and hands-on time should be considered for the selection of appropriate rapid ID and AST of PBC methods in different laboratory settings.

Germline PALB2 Mutation in High-Risk Chinese Breast and/or Ovarian Cancer Patients.

The prevalence of the PALB2 mutation in breast cancer varies across different ethnic groups; hence, it is of intense interest to evaluate the cancer risk and clinical association of the PALB2 mutation in Chinese breast and/or ovarian cancer patients. We performed sequencing with a 6-gene test panel (BRCA1, BRCA2, TP53, PTEN, PALB2, and CDH1) to identify the prevalence of the PALB2 germline mutation among 2631 patients with breast and/or ovarian cancer. In this cohort, 39 mutations were identified with 24 types of mutation variants, where the majority of the mutations were frame-shift mutations and resulted in early termination. We also identified seven novel PALB2 mutations. Most of the PALB2 mutation carriers had breast cancer (36, 92.3%) and were more likely to have family history of breast cancer (19, 48.7%). The majority of the breast tumors were invasive ductal carcinoma (NOS type) (34, 81.0%) and hormonal positive (ER: 32, 84.2%; PR: 23, 60.5%). Pathogenic mutations of PALB2 were found in 39 probands with a mutation frequency of 1.6% and 1% in breast cancer and ovarian cancer patients, respectively. PALB2 mutation carriers were more likely have hormonal positive tumors and were likely to have familial aggregation of breast cancer.

Characteristics and predictors of early hospital deaths in newly diagnosed APL: a 13-year population-wide study.

Despite therapeutic advances, early death (ED) remains a major factor curtailing survival of acute promyelocytic leukemia (APL). Studies examining factors that cause early death (ED; within 30 days of admission) and the correlation of survival with the timing of administration of all-trans retinoic acid (ATRA) and hemostatic parameters are scarce. We performed a cohort analysis of nonselect patients with newly diagnosed APL who presented to the health care system in Hong Kong, where oral arsenic trioxide was used. From 1 January 2007 to 30 April 2020, 358 patients (median age, 47 [1-97] years) with newly diagnosed APL were identified. ED occurred in 56 patients (16%): 11 (3%) died in the first 2 days after admission (intracranial hemorrhage [ICH], n = 6; APL-differentiation syndrome [APL-DS], n = 4; infection, n = 1); 22 (6%) died within 3 to 7 days (ICH, n = 12; APL-DS, n = 8; infections, n = 2), and 23 (6%) died within 8 to 30 days (ICH, n = 7; APL-DS, n = 11; infection, n = 5). Factors significantly associated with ED by multivariate analysis included male sex (P = .01); presenting leukocyte count ≥10 × 109/L (P = .03); fibrinogen <1.5 g/L (P = .02); and ATRA administration >24 hours after hospital admission (P < .001). After a median follow-up of 47 (0-166) months, the 5- and 10-year overall survival (OS) was 68.6% and 61.2%, respectively. Excluding EDs, the 5- and 10-year post-30-day OS improved to 81.3% and 72.5%. Early administration of ATRA (<24 hours) and vigorous correction of hemostatic abnormalities, including hypofibrinogenemia, are key to reducing ED.

A Case Report of Germline Compound Heterozygous Mutations in the BRCA1 Gene of an Ovarian and Breast Cancer Patient.

The germline carrier of the BRCA1 pathogenic mutation has been well proven to confer an increased risk of breast and ovarian cancer. Despite BRCA1 biallelic pathogenic mutations being extremely rare, they have been reported to be embryonically lethal or to cause Fanconi anemia (FA). Here we describe a patient who was a 48-year-old female identified with biallelic pathogenic mutations of the BRCA1 gene, with no or very subtle FA-features. She was diagnosed with ovarian cancer and breast cancer at the ages of 43 and 44 and had a strong family history of breast and gynecological cancers.

Mutation screening of germline TP53 mutations in high-risk Chinese breast cancer patients.

BACKGROUND: Germline TP53 mutations are associated with Li-Fraumeni syndrome, a severe and rare hereditary cancer syndrome. Despite the rarity of germline TP53 mutations, the clinical implication for mutation carriers and their families is significant. The risk management of TP53 germline mutation carriers is more stringent than BRCA carriers, and radiotherapy should be avoided when possible. METHODS: TP53 gene mutation screening was performed in 2538 Chinese breast cancer patients who tested negative for BRCA mutations. RESULTS: Twenty TP53 mutations were identified with high next-generation sequencing concerning for germline mutations in Chinese breast cancer families. The majorities of the TP53 carriers had early-onset, hormone receptor-positive breast cancer, and had strong family history of cancer. Among all, 11 patients carried a germline mutation and 6 of which were likely de novo germline mutations. In addition, 1 case was suspected to be induced by chemotherapy or radiation, as this patient had no significant family history of cancer and aberrant clonal expansion can commonly include TP53 mutations. Furthermore, we have identified one mosaic LFS case. Two novel mutations (c.524_547dup and c.529_546del) were identified in patients with early-onset. CONCLUSIONS: In view of the high lifetime risk of malignancy, identification of patients with germline TP53 mutations are important for clinicians to aid in accurate risk assessment and offer surveillance for patients and their families.

Genome Sequences of SARS-CoV-2 Strains Detected in Hong Kong.

We sequenced severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from deep throat saliva samples of three imported cases in Hong Kong by Nanopore sequencing. Epidemiological and clinical features of these coronavirus disease 2019 (COVID-19) cases were presented for genomic epidemiology studies.

Myeloproliferative neoplasms treated with hydroxyurea, pegylated interferon alpha-2A or ruxolitinib: clinicohematologic responses, quality-of-life changes and safety in the real-world setting.

Introduction: Real-world data of responses, quality-of-life (QOL) changes and adverse events in patients with myeloproliferative neoplasms (MPN) on conventional therapy (hydroxyurea ± anagrelide), pegylated interferon alpha-2A (PEG-IFNα-2A) or ruxolitinib are limited. Methods: We prospectively studied MPN patients receiving conventional therapy, PEG-IFNα-2A or ruxolitinib. Next-generation sequencing of 69 myeloid-related genes was performed. Clinicohematologic responses, adverse events, and QOL (determined by the Myeloproliferative Neoplasm Symptom Assessment Form Total Symptom Score, MPN-SAF TSS) were evaluated. Results: Seventy men and fifty-five women with polycythemia vera (PV) (N = 23), essential thrombocythemia (ET) (N = 56) and myelofibrosis (MF) (N = 46) were studied for a median of 36 (range: 19-42) months. In PV, responses were comparable for different modalities. CREBBP mutations were associated with inferior responses. In ET, PEG-IFNα-2A resulted in superior clinicohematologic complete responses (CHCR) (P = 0.045). In MF, superior overall response rates (ORR) were associated with ruxolintib (P = 0.018) and JAK2V617F mutation (P = 0.04). For the whole cohort, ruxolitinib led to rapid and sustained reduction in spleen size within the first 6 months, and significant improvement of QOL as reflected by reduction in MPN-SAF TSS (P < 0.001). Adverse events of grades 1-2 were observed in 44%, 62% and 20% of patients receiving conventional therapy, PEG-IFNα-2A and ruxolitinib respectively; and of grade 3-4 in 7% and 9% of patients receiving PEG-IFNα-2A and ruxolitinib. Conclusions: Conventional therapy, PEG-IFNα-2A and ruxolitinib induced responses in all MPN subtypes. PEG-IFNα-2A led to superior CHCR in ET; whereas ruxolitinib resulted in superior ORR in MF, and significant reduction in spleen size and improvement in QOL.

Clofarabine, cytarabine, and mitoxantrone in refractory/relapsed acute myeloid leukemia: High response rates and effective bridge to allogeneic hematopoietic stem cell transplantation.

Clofarabine is active in refractory/relapsed acute myeloid leukemia (AML). In this phase 2 study, we treated 18- to 65-year-old AML patients refractory to first-line 3 + 7 daunorubicin/cytarabine induction or relapsing after 3 + 7 induction and high-dose cytarabine consolidation, with clofarabine (30 mg/m2 /d, Days 1-5), cytarabine (750 mg/m2 /d, Days 1-5), and mitoxantrone (12 mg/m2 /d, Days 3-5) (CLAM). Patients achieving remission received up to two consolidation cycles of 50% CLAM, with eligible cases bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT). The mutational profile of a 69-gene panel was evaluated. Twenty-six men and 26 women at a median age of 46 (22-65) years were treated. The overall response rate after the first cycle of CLAM was 90.4% (complete remission, CR: 69.2%; CR with incomplete hematologic recovery, CRi: 21.2%). Twenty-two CR/CRi patients underwent allo-HSCT. The 2-year overall survival (OS), relapse-free survival (RFS), and event-free survival (EFS) were 65.8%, 45.7%, and 40.2%, respectively. Multivariate analyses showed that superior OS was associated with CR after CLAM (P = .005) and allo-HSCT (P = .005), and superior RFS and EFS were associated with allo-HSCT (P < .001). Remarkably, CR after CLAM and allo-HSCT resulted in 2-year OS of 84.3% and 90%, respectively. Karyotypic aberrations and genetic mutations did not influence responses or survivals. Grade 3/4 neutropenia/thrombocytopenia and grade 3 febrile neutropenia occurred in all cases. Other nonhematologic toxicities were mild and uncommon. There was no treatment-related mortality and the performance of allo-HSCT was not compromised. Clofarabine, cytarabine, and mitoxantrone was highly effective and safe in refractory/relapsed AML. This study was registered at ClinicalTrials.gov (NCT02686593).

Germline Mutation in 1338 BRCA-Negative Chinese Hereditary Breast and/or Ovarian Cancer Patients: Clinical Testing with a Multigene Test Panel.

Differences in the mutation spectrum across ethnicities suggest the importance of identifying genes in addition to common high penetrant genes to estimate the associated breast cancer risk in China. A total of 1338 high-risk breast cancer patients who tested negative for germline BRCA1, BRCA2, TP53, and PTEN mutations between 2007 and 2017 were selected from the Hong Kong Hereditary Breast Cancer Family Registry. Patient samples were subjected to next-generation DNA sequencing using a multigene panel (Color Genomics). All detected pathogenic variants were validated by bidirectional DNA sequencing. The sequencing data were coanalyzed by a bioinformatics pipeline developed in-house. Sixty-one pathogenic variants (4.6%) were identified in this cohort in 11 cancer predisposition genes. Most carriers (77.1%) had early onset of breast cancer (age <45 years), 32.8% had family members with breast cancer, and 11.5% had triple-negative breast cancer. The most common mutated genes were PALB2 (1.4%), RAD51D (0.8%), and ATM (0.8%). A total of 612 variants of unknown significance were identified in 494 patients, and 87.4% of the variants of unknown significance were missense mutations. Pathogenic variants in cancer predisposition genes beyond BRCA1, BRCA2, TP53, and PTEN were detected in an additional 4.6% of patients using the multigene panel. PALB2 (1.4%) and RAD51D (0.8%) were the most commonly mutated genes in patients who tested mutation negative by a four-gene panel.

Sorafenib and omacetaxine mepesuccinate as a safe and effective treatment for acute myeloid leukemia carrying internal tandem duplication of Fms-like tyrosine kinase 3.

BACKGROUND: Omacetaxine mepesuccinate (OME) has antileukemic effects against acute myeloid leukemia (AML) carrying an internal tandem duplication of Fms-like tyrosine kinase 3 (FLT3-ITD). A phase 2 clinical trial was conducted to evaluate a combination treatment of sorafenib and omacetaxine mepesuccinate (SOME). METHODS: Relapsed or refractory (R/R) or newly diagnosed patients were treated with sorafenib (200-400 mg twice daily) and OME (2 mg daily) for 7 (first course) or 5 days (second course onward) every 21 days until disease progression or allogeneic hematopoietic stem cell transplantation (HSCT). The primary endpoint was composite complete remission, which was defined as complete remission (CR) plus complete remission with incomplete hematologic recovery (CRi). Secondary endpoints were leukemia-free survival (LFS) and overall survival (OS). RESULTS: Thirty-nine R/R patients and 5 newly diagnosed patients were recruited. Among the R/R patients, 28 achieved CR or CRi. Two patients showed partial remission, and 9 patients did not respond. Among the 5 newly diagnosed patients, 4 achieved CR, and 1 achieved CRi. The median LFS and OS were 5.6 and 10.9 months, respectively. Prior Fms-like tyrosine kinase 3 (FLT3) inhibitor exposure (P = .007), 2 or more inductions (P = .001), and coexisting IDH2 (P = .008) and RUNX1 mutations (P = .003) were associated with lower CR/CRi rates. HSCT consolidation and deep molecular responses (defined as an FLT3-ITD variant allelic frequency [VAF] ≤ 0.1% or a nucleophosmin 1 [NPM1] mutant VAF ≤ 0.01%) were associated with better OS and LFS. Prior FLT3 inhibitor exposure and 2 or more inductions were associated with inferior LFS. CONCLUSIONS: SOME was safe and effective for R/R and newly diagnosed FLT3-ITD AML.

Rapid detection of chromosomal translocation and precise breakpoint characterization in acute myeloid leukemia by nanopore long-read sequencing.

Detection of chromosomal translocation is a key component in diagnosis and management of acute myeloid leukemia (AML). Targeted RNA next-generation sequencing (NGS) is emerging as a powerful and clinically practical tool, but it depends on expression of RNA transcript from the underlying DNA translocation. Here, we show the clinical utility of nanopore long-read sequencing in rapidly detecting DNA translocation with exact breakpoints. In a newly diagnosed patient with AML, conventional karyotyping showed translocation t(10;12)(q22;p13) but RNA NGS detected NUP98-NSD1 fusion transcripts from a known cryptic translocation t(5;11)(q35;p15). Rapid PCR-free nanopore whole-genome sequencing yielded a 26,194 bp sequencing read and revealed the t(10;12) breakpoint to be DUSP13 and GRIN2B in head-to-head configuration. This translocation was then classified as a passenger structural variant. The sequencing also yielded a 20,709 bp sequencing read and revealed the t(5;11) breakpoint of the driver NUP98-NSD1 fusion. The identified DNA breakpoints also served as markers for molecular monitoring, in addition to fusion transcript expression by digital PCR and sequence mutations by NGS. We illustrate that third-generation nanopore sequencing is a simple and low-cost workflow for DNA translocation detection.

Distinct mutation spectrum, clinical outcome and therapeutic responses of typical complex/monosomy karyotype acute myeloid leukemia carrying TP53 mutations.

The present study aimed to define a subtype of complex/monosomal karyotype (CK/MK) acute myeloid leukemia (AML) by its distinct clinical features, p53 signaling and responses to p53 targeting agents. Ninety-eight young adults (range: 21-60 years; median: 49 years) with CK/MK AML were studied. They received standard induction, consolidation and allogeneic hematopoietic stem cell transplantation from siblings or matched unrelated donors if available. Chromosomal abnormalities most commonly affected chromosome 5 (30%), 7 (22%) and 17 (21%). Next generation sequencing of a 54-myeloid gene panel were available in 76 patients. Tumor protein 53 (TP53) mutations were most common (49%) and associated with the presence of -5/5q- (P < .001) and -17/17p- (P < .001), but not -7/7q- (P = .370). This "typical" CK/MK AML subtype was associated with significantly lower presenting white cell counts, higher number of karyotypic abnormalities, and inferior leukemia-free and overall survivals, compared with CK/MK AML without the typical features. Blood or bone marrow samples from typical CK/MK AML patients showed defective p53 signaling upon induction by etoposide. In vitro drug sensitivity analysis showed that they were sensitive to APR-246 that targeted mutant p53, but resistant to MDM2 antagonist MI-77301. Novel therapeutic strategies targeting TP53 mutations in CK/MK AML should be developed and tested in clinical trials.